Nucleic Acids Research

Deoxyoligonucleotide probes

The deoxyoligonucleotide probes were custom synthesized by Kurabo Co. (Osaka) and Sawady technology (Tokyo). The sequences are listed below, in which the hairpin sequence (underlined) and mismatched bases (bold) are emphasized. TRI-20-63, 5[prime]-GTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTGCGGCCGCGGTTTCCGCGGCCGC-3[prime]; TRI-20-62, 5[prime]-GTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTGCGGCCGCGGTTTCCGCGGCCG-3[prime]; TRI-20-64, 5[prime]-GTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTGCGGCCGCGGTTTCCGCGGCCGCG-3[prime]; TRI-20-50, 5[prime]-GTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTGCGGCCGCGG-3[prime]; TRI-10-63, 5[prime]-ATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGCGGCCGCGGTTTCCGCGGCCGC-3[prime]; TRI-20-63M1, 5[prime]-GTCATGCCATCCGTAAGATGATTTTCTGTGACTGGTGAGTGCGGCCGCGGTTTCCGCGGCCGC-3[prime]; TRI-20-63M2, 5[prime]-GTCATGCCATACGTAAGATGATTTTCTGTGACTGGTGAGTGCGGCCGCGGTTTCCGCGGCCGC-3[prime]; TRI-20-63M3, 5[prime]-GTCATGCCATACGTAAGATGATTTTCTGTGCCTGGTGAGTGCGGCCGCGGTTTCCGCGGCCGC-3[prime]; TRI-20-63M4, 5[prime]-GTCATGCCATACGTACGATGATTTTCTGTGCCTGGTGAGTGCGGCCGCGGTTTCCGCGGCCGC-3[prime]; TRI-20-63M5, 5[prime]-GTCATGCCATACGTACGATGATTTTATGTGCCTGGTGAGTGCGGCCGCGGTTTCCGCGGCCGC-3[prime]; TRI-21-63, 5[prime]-GTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGGCGGCCGCGGTTTCCGCGGCCGC-3[prime]; TRI-22-63, 5[prime]-TCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGGCGGCCGCGGTTTCCGCGGCCGC-3[prime]; TRI-PA20-63, 5[prime]-GCGGCCGCGGTTTCCGCGGCCGCACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGAC-3[prime]; FTZ-1-63, 5[prime]-CCTACGGTGCCCAGGACATTTTGGGCACAAGGACGAGTGCGCGGCCGCGGTTTCCGCGGCCGC-3[prime]; P53-1-63, 5[prime]-CAAGATGTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGGCGGCCGCGGTTTCCGCGGCCGC-3[prime]; P53-3-63, 5[prime]-GGGTTGGGGTCGGGGTGGTGGCCTGCCCTTCCAATGGATCGCGGCCGCGGTTTCCGCGGCCGC-3[prime].

DNA purification

pBluescript DNA was extracted by alkaline procedure (10) from Escherichia coli (XL1-Blue) which had been cultured in Terrific broth (10) containing ampicillin (50 µg/ml). DNA in the alkaline supernatant was further purified by CsCl centrifugation(85 000 r.p.m. for 16 h), which was followed by Sephacryl S-400 chromatography after dialysis against TE buffer. The DNA was then treated with phenol and chloroform, then precipitated with ethanol and dissolved in TE buffer at a DNA concentration of 200 µg/ml. Drosophila and human DNA were purchased from Promega.

Direct probing

For direct probing of pBluescript DNA, deoxyoligonucleotide probes (5 ng) labeled at the 5[prime]-terminus with ³²P using T4 polynucleotide kinase (Megalabel; Takara) were incubated in a reaction mixture (5 µl) which contained recA protein (0.48 µg; Epicentre Technologies or Pharmacia Biotech), ATP-[gamma]-S (4.8 mM; Sigma), magnesium acetate (2.5 mM) and Tris-acetate (30 mM, pH 7.2), for 15 min at 37°C. A ScaI digest of double-stranded DNA (100 ng, pBluescript DNA; Stratagene) in a buffer (5 µl) containing magnesium acetate (22.5 mM), ATP-[gamma]-S (4.8 mM) and Tris-acetate (30 mM) was then added and incubated for another 30 min. Heat-stable DNA ligase (20 U, Ampligase; Epicentre Technologies) in 10 µl of a buffer (10× RXN buffer diluted 5-fold with redistilled water, provided by the supplier) was then added and the reaction mixture was incubated for 15 min at 65°C. The reaction was terminated by adding 1.8 µl of EDTA (110 mM)/SDS (5.6%) solution, which was followed by incubation for 15 min at 37°C after addition of proteinase K (1 µl, 20 mg/ml). DNA was precipitated with ethanol and dissolved in TE buffer (10 µl). The samples were then subjected to neutral or alkaline (0.05 M NaOH, 1 mM EDTA) agarose gel (0.8%) electrophoresis (120 V, 2 h or 20 V, 5 h, respectively), the gels stained with ethidium bromide and autoradiographed (5 h at -80°C) using Fuji X-ray film (RX).

For probing of specific DNA fragments in the fruit fly (Drosophila melanogaster) or human genomes, higher concentrations of probes, target DNA and recA (25 ng, 20 µg and 7.84 µg, respectively) were employed and excess unreacted labeled oligonucleotide probes were removed before electrophoresis through a Sephacryl S-400 spin column to reduce background noise (legend to Fig. 7).

Principle of the procedure

The procedure is outlined diagrammatically in Figure 1. The oligonucleotide probe specifically designed for this purpose consists of two parts; a probing sequence complementary to the terminal sequence of target DNA and a universal hairpin structure which makes it possible to covalently link the probe to the target DNA. In the first step, the homologous sequence present at the terminus of target DNA is recognized and one of the strands is displaced by the complementary sequence of the oligonucleotide probe through a recA protein-mediated reaction. Second, the probe is covalently attached to the target DNA by DNA ligase. The hairpin-like structure of the probe brings the 3[prime]-terminus of the probe oligonucleotide into close stereochemical proximity to the 5[prime]-terminus of the complementary strand of target DNA for ligation. In the actual process, the labeled oligonucleotide probe was preincubated with recA protein, then incubated with target DNA to form a probe-target DNA-recA protein complex with a partial triplex structure. DNA ligase is then added to covalently attach the probe to the target DNA. After this reaction, the proteins are removed and the products can then be subjected to further analyses such as gel electrophoresis to detect hybridized DNA fragments. The hairpin probes can also be designed to probe the opposite complementary sequence at the same terminus, other terminal sequences of the same fragment, or both (below).

Figure 1. Diagrammatic outline of direct probing of specific base sequences in double-stranded DNA by hairpin-like oligonucleotide probes. Target DNA and deoxyoligonucleotide probe are shown in blue and red, respectively. Conversion between the two structures (III and IV) is highly speculative and therefore IV is shown in parentheses.

Direct probing

In Figure 2, we show the results of a model experiment in which a double-stranded DNA [pBluescript SK(-), a derivative of pBR322] was probed by a 5[prime]-labeled hairpin deoxyoligonucleotide (TRI-20-63), which consists of a 40mer base sequence complementary to the 3[prime]-terminal sequence of a ScaI digest of Bluescript (a derivative of pBR322; Stratagene) DNA attached to a 23mer hairpin-like structure which in turn consists of a 20mer base paired (10 bp) region plus a 3mer hinged region (Fig. 1). The probe was first incubated with recA protein (15 min at 37°C), mixed with target DNA and incubated further (30 min at 37°C). A heat-stable ligase (Ampligase) was added and incubation was continued for 15 min at 65°C. The recA protein and ligase in the reaction mixture were then removed by proteinase K (or phenol) treatment and the samples were autoradiographed after electrophoresis. As seen in Figure 2A, only when both recA protein and DNA ligase were present in the reaction was a signal detected at the position of 3 kb where double-stranded Bluescript DNA is expected to migrate. The signal was still present even after the sample was subjected to alkaline electrophoresis (Fig. 2B), indicating that the probe was covalently attached to the target DNA molecule. Due to elimination of the DNA dissociation steps and use of recA protein as well as heat-resistant DNA ligase, the entire reaction can be completed in just 1 h, only a fraction of the time required for similar procedures currently in use. Because we employed recA protein (and DNA ligase), complex formation between probe and target DNA was quite efficient as we estimate that >60% of target DNA was associated with probe (measured with a Fuji BAS 2000 image analyzer) under the conditions we employed (data not shown). As expected, signal intensities increased proportionally as the quantities of target DNA were increased (data not shown), suggesting that simple quantitation of target DNA is possible as it is in Southern hybridization.

Figure 2. Direct probing of pBluescript DNA: effects of recA protein and DNA ligase. A deoxyoligonucleotide probe (TRI-20-63, 5 ng) labeled at the 5[prime]-terminus with ³²P by T4 polynucleotide kinase (Megalabel; Takara) was incubated in a reaction mixture (5 µl) which contained recA protein (0.48 µg; Pharmacia Biotech), ATP-[gamma]-S (4.8 mM; Sigma), magnesium acetate (2.5 mM) and Tris-acetate (30 mM, pH 7.2), for 15 min at 37°C. A ScaI digest of double-stranded DNA (100 ng, pBluescript DNA; Stratagene) in a buffer (5 µl) containing ATP-[gamma]-S (4.8 mM), magnesium acetate (22.5 mM) and Tris-acetate (30 mM) was then added and further incubated for another 30 min. Heat-stable DNA ligase (20 U, Ampligase; Epicentre Technologies) in 10 µl of a buffer (10× RXN buffer diluted 5-fold with redistilled water) provided by the supplier was then added and the reaction mixture was incubated for 15 min at 65°C. The reaction was terminated by adding 1.8 µl of EDTA (110 mM)/SDS (5.6%) solution which was followed by incubation for 15 min at 37°C after addition of proteinase K (1 µl, 20 mg/ml). DNA was precipitated with ethanol and dissolved in TE buffer (10 µl). The samples were then subjected to neutral or alkaline (0.05 M NaOH, 1 mM EDTA) agarose gel (0.8%) electrophoresis (120 V, 2 h or 20 V, 5 h, respectively), the gels stained with ethidium bromide and autoradiographed (5 h at -80°C) using Fuji X-ray film (RX). (A) Autoradiographic patterns after neutral agarose gel electrophoresis. (B) Autoradiographic patterns after alkaline agarose gel electrophoresis. (C) Ethidium bromide staining patterns after neutral agarose gel electrophoresis. (A-C) Lane 1, without recA protein and DNA ligase; lane 2, without DNA ligase; lane 3, without recA protein; lane 4; control (complete); lane M, DNA size markers([lambda] HindIII digests) with their approximate molecular sizes in kb. Details in Materials and Methods.

Probing by various hairpin probes

Figure 3 shows the results of probing the same target DNA with various hairpin probes. First, no signals were detected when the target DNA was pretreated before the reaction with bacterial alkaline phosphatase to eliminate the terminal (5[prime]) phosphate which is necessary for ligation of the probe to target DNA (lane 2). Likewise, probes that were one base short (TRI-20-62) or had one extra base (TRI-20-64) at the 3[prime]-terminus did not produce a signal (lanes 3 and 4, respectively). A DNA probe (TRI-20-50) similar to the standard probe (TRI-20-63) but with a DNA sequence which does not form a hairpin structure also failed to produce the signal (lane 5). Another probe (TRI-10-63), which carries a 40mer sequence complementary to those located within (nt 2487-2526) the ScaI fragment rather than to the terminal sequence as in TRI-20-63, also did not produce a signal (lane 6). Furthermore, no signals were detected when EcoRV-digested Bluescript DNA, in which the target sequence is located inside, not at the terminus, was employed as a target DNA with the standard probe (TRI-20-63) (lane 7). From these results, it is clear that the signal is produced only under conditions in which ligation can be achieved between the 3[prime]-terminus of the hairpin probe and 5[prime]-terminus of the target DNA.

Figure 3. Direct probing of pBluescript DNA with various deoxyoligonucleotide probes. ³²P-labeled deoxyoligonucleotide probes (sequences below and in Materials and Methods) were incubated with recA protein (0.48 µg), further incubated with restriction enzyme (ScaI or EcoRV)-digested or dephosphorylated pBluescript DNA (100 ng) and subjected to ligation with ligase as described in the legend to Figure 2. After termination of the reaction and proteinase K treatment, samples were electrophoresed (neutral) and autoradiographed. The following combinations of probe and target DNA were employed. Lane 1, TRI-20-63 (probe) and ScaI-digested pBluescript DNA (target); lane 2, TRI-20-63 (probe) and ScaI-digested and alkaline phosphatase-treated pBluescript DNA (target); lane 3, TRI-20-62 (probe) and ScaI-digested pBluescript DNA (target); lane 4, TRI-20-64 (probe) and ScaI-digested pBluescript DNA (target); lane 5, TRI-20-50 (probe) and ScaI-digested pBluescript DNA (target); lane 6, TRI-10-63 (probe) and ScaI-digested pBluescript DNA (target); lane 7, TRI-20-63 (probe) and EcoRV-digested pBluescript DNA (target). For alkaline phosphatase treatment (lane 2), a ScaI digest of pBluescript DNA (5 µg) was treated with bacterial alkaline phosphatase (0.45 U; Toyobo) for 16 h at 37°C in a reaction mixture as suggested by the manufacturer. After treating the mixture with phenol, DNA was precipitated with ethanol and dissolved in TE buffer at a concentration of 200 µg/ml. DNA size markers ([lambda] HindIII digests) are shown in lane M with their approximate molecular sizes in kb. Details in Materials and Methods.

In a separate series of experiments, fidelity of the recA protein-mediated reaction in recognizing homologous sequences in target DNA was examined using hairpin probes with varying numbers of mismatched bases. As seen in Figure 4, hairpin probes with one or two mismatched bases in the 40mer complementary sequences could be ligated to target DNA without any appreciable loss of efficiency (lanes 2 and 3) compared with control probe (lane 1). The efficiency, however, was significantly reduced when three or four mismatched bases were introduced into the probe (lane 4 and 5) and no signal was detected when five mismatched bases were introduced (lane 6). Thus, recA protein recognizes homologous base sequences with at least two mismatched bases in the 40mer terminal sequence with seemingly equal efficiency as it does completely matched sequences.

Figure 4. Effect of mismatched bases on direct probing. ScaI-digested pBluescript DNA was probed with ³²P-labeled deoxyoligonucleotides with increasing numbers of mismatched bases (sequences in Materials and Methods) as described in the legend to Figure 2 except that unreacted probes were removed by a Sephacryl S-400 column before ethanol precipitation. Lane 1, control probe (TRI-20-63); lane 2, one base mismatched probe (TRI-20-63M1); lane 3, two base mismatched probe (TRI-20-63M2); lane 4, three base mismatched probe (TRI-20-63M3); lane 5, four base mismatched probe (TRI-20-63M4); lane 6, five base mismatched probe (TRI-20-63M5). DNA size markers ([lambda] HindIII digests) are shown in lane M with their approximate molecular size in kb. Details in Materials and Methods.

In the model experiments described above, we employed target DNA with blunt ends (ScaI-treated Bluescript DNA). As seen in Figure 5, target DNA molecules with other forms of termini such as 5[prime] protruding (AflII-treated DNA) (lane 1) or 3[prime] protruding (AlwNI-treated DNA) (lane 3) can also serve as targets for ligation with almost equal efficiencies as observed for DNA with blunt ends (lane 2). This indicates that DNA fragments produced by various restriction enzymes can be subjected to direct probing.

Figure 5. Direct probing of double-stranded target DNA carrying different terminal structures. pBluescript DNA was digested with AflIII, ScaI or AlwNI which produces DNA fragments with 5[prime] protruding (four bases), blunt and 3[prime] protruding (three bases) termini, respectively. Each digest was probed by ³²P-labeled deoxyoligonucleotide probes, TRI-21-63 for AflIII digest, TRI-20-63 for ScaI digest and TRI-22-63 for AlwNI digest, as described in the legend to Figure 2 except that unreacted probes were removed by a Sephacryl S-400 column before ethanol precipitation. Probe sequences in Materials and Methods. Lane 1, AflIII digest probed by TRI-21-63; lane 2, ScaI digest probed by TRI-20-63; lane 3, AlwNI digest probed by TRI-22-63. DNA size markers ([lambda] HindIII digests) are shown in lane M with their approximate molecular size in kb. Details in Materials and Methods.

Next, probing the complementary sequence of the opposite strand at the same terminus was examined. As seen in Figure 6, ligation of a hairpin probe (TRI-PA20-63) to the 3[prime]-terminus of the opposite strand was equally effective (lane 2) as that to the 5[prime]-terminus of the target DNA (lane 1) with TRI-20-63 as shown above.

Figure 6. Direct probing of sequences of the opposite strand at the same terminus. ³²P-labeled deoxyoligonucleotide probes, TRI-20-63 and TRI-PA20-63, which carry the complementary sequences of the opposite strand of terminal sequences probed by TRI-20-63 (sequence in Materials and Methods), were incubated with recA protein (0.48 µg), further incubated with ScaI-digested pBluescript DNA (100 ng) and subjected to ligation and electrophoresis as described in the legend to Figure 2 except that unreacted probes were removed by a Sephacryl S-400 column before ethanol precipitation. Lane 1, probed by TRI-20-63; lane 2, probed by TRI-PA20-63. DNA size markers ([lambda] HindIII digests) are shown in lane M with their approximate molecular size in kb. Details in Materials and Methods.

Figure 7. Direct probing of genes in Drosophila (ftz) and human (p53) genomes. (A) Drosophila melanogaster DNA (Promega) was digested with FspI (47 U; NEB) or EcoRV (48 U; Toyobo) for 16 h at 37°C in a 200 µl reaction mixture as described in the manufacturer's instructions. After phenol extraction, DNA was precipitated with ethanol and dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) at a concentration of 4 µg/µl. The DNA (20 µg; Promega) was incubated with 25 ng of a ³²P-labeled hairpin probe (FTZ-1-63, sequence in Materials and Methods) for the FspI fragment of the ftz gene (11) which had been incubated with recA protein (7.84 µg). The sample was subjected to a ligation reaction with Ampligase, as described in the legend to Figure 2 except that the ligation was performed for 16 h at 55°C. After termination of the reaction and proteinase K treatment (legend to Fig. 2), unreacted probe was removed by a Sephacryl S-400 column. The samples (concentrated to 20 µl) were then electrophoresed (neutral) and autoradiographed (legend to Fig. 2). (Left) Lane 1, FspI-digested DNA; lane 2, EcoRV-digested DNA. The position of the band is indicated by an arrow. (Right) Ethidium bromide staining pattern after electrophoresis. (B) Human DNA (Promega) was digested with BamHI (30 U; NEB) and PvuII (30 U; NEB) or EcoRV (30 U; NEB) for 16 h at 37°C in a 200 µl reaction mixture and purified as described above. The BamHI and PvuII double-digested or EcoRV-digested DNA (20 µg, Promega) was subjected to direct probing as was the FspI fragment in Drosophila DNA (above) except that (i) two labeled hairpin probes (25 ng each) in which the 40mer sequences are complementary to the 3[prime]-terminal (P53-1-63) and 5[prime]-terminal sequence (P53-3-63) of the p53 fragment were used simultaneously, (ii) the ligase reaction was performed at 60°C and (iii) unreacted labeled oligonucleotides were removed by three repeated spin columns before electrophoresis to reduce background noise. (Left) Lane 1. BamHI and PvuII double-digested DNA; lane 2, EcoRV-digested DNA. The position of the band is indicated by an arrow. (Right) Ethidium bromide staining pattern after electrophoresis. Details in Materials and Methods.

It should be noted that despite many attempts we failed to covalently attach the hairpin probe to the termini of complementary single-stranded DNA after conventional hybridization of probe to target DNA. The reason for the difficulty in ligating the probe to single-stranded DNA and not to double-stranded DNA as described above is not fully understood at the present time but is currently under investigation.

Probing a specific sequence in genomic DNA

We examined whether a specific sequence in genomic DNA can be directly probed by hairpin probes. First, one of the homeobox genes (fushi tarazu, ftz) in D.melanogaster (11), which is present as a single copy in a genome of ~180 Mb, was subjected to this investigation. Whole Drosophila genomic DNA was digested with FspI, which produces a 1753 bp ftz gene fragment, and the digest was subjected to direct probing using a hairpin probe (FTZ-1-63) in which a 40mer sequence is complementary to the 3[prime]-terminal sequence of the FspI fragment. As seen in Figure 7A, a band of ~1.75 kb corresponding to the FspI fragment of the ftz gene in size was observed (lane 1, indicated by an arrow). No signals were detected when EcoRV-digested Drosophila DNA in which the sequence corresponding to probe is not located at the 3[prime]-terminus was used as target DNA (lane 2).

Similar experiments were performed to probe a single copy DNA fragment (p53 gene) in the human genome (~3000 Mb), which is 17 times more complex than the Drosophila genome. Human genomic DNA was digested with BamHI and PvuII, which produces a 1312 bp p53 gene fragment (12), and the digest was subjected to direct simultaneous probing with two hairpin probes in which the respective 40mer sequences are complementary to the 3[prime]-terminal (P53-1-63) and 5[prime]- terminal sequences (P53-3-63) of the p53 fragment. As seen in Figure 7B, after eliminating most of the unreacted labeled oligonucleotides before electrophoresis (legend to Fig. 7) to reduce background noise and thus to detect a single gene fragment in a complex genome by an oligonucleotide probe, we were able to detect a band corresponding to the targeted fragment (lane 1, indicated by an arrow). No signals were detected with a control DNA sample (EcoRV-digested DNA), in which the sequences corresponding to the probes are not located at either terminus (lane 2). These results strongly suggest that direct probing of specific base sequences can be achieved even in highly complex genomes such as the Drosophila and human genomes.

DISCUSSION

In this paper we have described a novel procedure which can be applied to probing of specific DNA sequences. By employing a hairpin-like probe, it has become possible to covalently attach probe DNA directly to double-stranded target DNA. Elimination of DNA dissociation and subsequent hybridization (and washing) have made the entire process of probing streamlined and efficient, achievable within a fraction of the time required for similar procedures currently in use such as Southern hybridization, while maintaining high probing efficiency. In addition, direct probing has proven to have other more specific advantages over conventional procedures. For example, when one probes a single sample with multiple probes, repeated hybridization (rehybridization) is no longer necessary as probing can be carried out simultaneously in separate tubes for separate probes and samples can be electrophoresed in parallel. This may be particularly advantageous in non-PCR-based polymorphism analysis using multiple probes. Our novel procedure makes it easier to detect a single copy DNA fragment in highly complex genomes using oligonucleotide probes than with current procedures, as shown above in probing the human p53 gene fragment. We attributed this to (i) the high efficiency of recA protein in finding complementary sequences and (ii) removal of unreacted labeled probes before electrophoresis, both of which are unique to this procedure.

In the procedure described, base sequences located at the terminus of the target DNA are recognized and only sequences in that region can be probed. Because of this feature, ambiguous hybridization products derived from illegitimate hybridization with similar but not identical sequences at other locations, which are often encountered in current procedures and require re-examination by other means, should essentially be eliminated. On the other hand, since only sequences at the termini of restriction fragments can be recognized, one may encounter problems when sequences to be recognized cannot be placed at the termini for some reason or when the exact location of the sequence is not known.

The unique characteristics of the reported procedure seem to have virtually eliminated the anticipated obstacles for construction of fully automated probing equipment, which has yet to be realized despite a great potential need. Needless to say, this procedure could be exploited even more effectively by combining it with other molecular biological techniques such as PCR, depending upon the purposes of the experiments involved.

ACKNOWLEDGEMENTS

We thank Drs Y. Shigemori, K. Okumura and T. Yamamoto for their helpful discussions. This work was supported by the R & D Project of the Innovative Technology for the Earth Program which is sponsored by NEDO (New Energy and Industrial Technology Development Organization).

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*To whom correspondence should be addressed. Tel: +81 438 52 3944; Fax: +81 438 52 3911; Email: oishi@kazusa.or.jp

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